Composition of HMB and ATP and methods of use

ABSTRACT

The present invention provides a composition comprising HMB and ATP. Methods of administering HMB and ATP to an animal are also described. HMB and ATP are administered to increase power and strength. The combination of HMB and ATP together has a synergistic effect, which results in a surprising and unexpected level of improvement in power and strength. HMB and ATP are also administered to increase lean body mass and muscle hypertrophy and to prevent typical declines in performance that are characteristic of overreaching.

The present application claims priority to U.S. Patent Application Ser.No. 61/698,919, filed Sep. 10, 2012, which is incorporated herein in itsentirety by this reference.

BACKGROUND OF THE INVENTION

Field

The present invention relates to a composition comprisingβ-hydroxy-β-methylbutyrate (HMB) and adenosine-5′-triphosphate (ATP),and methods of using a combination of HMB and ATP to improve strengthand power, improve muscle mass and prevent or lessen typical declines inperformance characteristic of overreaching.

Background

HMB

The only product of leucine metabolism is ketoisocaproate (KIC). A minorproduct of KIC metabolism is β-hydroxy-β-methylbutyrate (HMB). HMB hasbeen found to be useful within the context of a variety of applications.Specifically, in U.S. Pat. No. 5,360,613 (Nissen), HMB is described asuseful for reducing blood levels of total cholesterol and low-densitylipoprotein cholesterol. In U.S. Pat. No. 5,348,979 (Nissen et al.), HMBis described as useful for promoting nitrogen retention in humans. U.S.Pat. No. 5,028,440 (Nissen) discusses the usefulness of HMB to increaselean tissue development in animals. Also, in U.S. Pat. No. 4,992,470(Nissen), HMB is described as effective in enhancing the immune responseof mammals. U.S. Pat. No. 6,031,000 (Nissen et al.) describes use of HMBand at least one amino acid to treat disease-associated wasting.

HMB is an active metabolite of the amino acid leucine. The use of HMB tosuppress proteolysis originates from the observations that leucine hasprotein-sparing characteristics. The essential amino acid leucine caneither be used for protein synthesis or transaminated to the α-ketoacid(α-ketoisocaproate, KIC). In one pathway, KIC can be oxidized to HMB.Approximately 5% of leucine oxidation proceeds via the second pathway.HMB is superior to leucine in enhancing muscle mass and strength. Theoptimal effects of HMB can be achieved at 3.0 grams per day, or 0.038g/kg of body weight per day, while those of leucine require over 30.0grams per day.

Once produced or ingested, HMB appears to have two fates. The first fateis simple excretion in urine. After HMB is fed, urine concentrationsincrease, resulting in an approximate 20-50% loss of HMB to urine.Another fate relates to the activation of HMB to HMB-CoA. Once convertedto HMB-CoA, further metabolism may occur, either dehydration of HMB-CoAto MC-CoA, or a direct conversion of HMB-CoA to HMG-CoA, which providessubstrates for intracellular cholesterol synthesis. Several studies haveshown that HMB is incorporated into the cholesterol synthetic pathwayand could be a source for new cell membranes that are used for theregeneration of damaged cell membranes. Human studies have shown thatmuscle damage following intense exercise, measured by elevated plasmaCPK (creatine phosphokinase), is reduced with HMB supplementation withinthe first 48 hrs. The protective effect of HMB lasts up to three weekswith continued daily use. Numerous studies have shown an effective doseof HMB to be 3.0 grams per day as CaHMB (calcium HMB) (˜38 mg/kg bodyweight-day⁻¹). This dosage increases muscle mass and strength gainsassociated with resistance training, while minimizing muscle damageassociated with strenuous exercise (34) (4, 23, 26). HMB has been testedfor safety, showing no side effects in healthy young or old adults. HMBin combination with L-arginine and L-glutamine has also been shown to besafe when supplemented to AIDS and cancer patients.

Recently, HMB free acid, a new delivery form of HMB, has been developed.This new delivery form has been shown to be absorbed quicker and havegreater tissue clearance than CaHMB. The new delivery form is describedin U.S. Patent Publication Serial No. 20120053240 which is hereinincorporated by reference in its entirety.

ATP

Adenosine-5′-triphosphate (ATP) has long been known as the chemicalenergy source for tissues including muscle (19). Intracellular ATPconcentrations (1-10 mM) are quite high in contrast to extracellularconcentrations (10-100 nM) and therefore release of ATP from cells suchas erythrocytes and muscle is strictly controlled. More recentlyextracellular effects of ATP, acting through purinergic receptors foundin most cell types, have been elicited (20). Several extracellularphysiological functions of ATP have been described includingvasodilation (21), reduced pain perception (22), and as aneurotransmission cotransmitter (23, 24). Importantly, small andtransient increases in vascular ATP in muscle can cause vasodilation andan increase in blood flow to the muscle (25). Therefore, if ATPincreases blood flow to muscle, especially during periods of strenuousresistance training, substrate availability would be improved andremoval of metabolic waste products would be better facilitated. Elliset al recently reviewed the studies supporting the role of ATP inincreasing muscle blood flow through purinergic signaling andneurotransmission (25).

ATP has been shown to have an inotrophic effect ATP on cardiac muscle(26, 27). Another study supporting systemic effects of ATP demonstratedthat oral administration of ATP to rabbits for 14 days resulted in areduction in peripheral vascular resistance, improvement of cardiacoutput, reduction of lung resistance, and increased arterial PaO₂ (28).

Adenosine, resulting from the degradation of ATP, may also act as asignaling agent through purinergic receptors (29) or may be degraded byadenosine deaminase (30). Adenosine acting through purinergic receptorscan essentially mimic the effects of ATP (29). Adenosine infusion intomuscle results in increased nitric oxide formation and similar vasculareffects as seen with ATP infusion (31).

Fatigue resistance in repeated high intensity bouts of exercise is amuch sought after attribute in athletics. This is true for bothaugmentation of training volume, as well as sustained force and poweroutput in intermittent sports such as hockey. During fatiguingcontractions acute adaptations in blood flow occur to stave off declinesin force generating capacity (40, 45). There is a tight coupling betweenoxygen demand in skeletal muscle and increases in blood flow (45).Research suggests that it is red blood cells that regulate this responseby acting as “oxygen sensors” (45). ATP is carried in red blood cellsand when oxygen is low in a working muscle region, the red blood celldeforms resulting in a cascade of events which lead to ATP release andbinding to endothelial cells in smooth muscle (43). Binding results insmooth muscle relaxation and subsequent increases in blood flow,nutrient and oxygen delivery (43). Specifically, extracellular ATPdirectly promotes the increased synthesis and release of nitric oxide(NO) and prostacyclin (PGl₂) within skeletal muscle and thereforedirectly affects tissue vasodilation and blood flow (31). This issupported by research suggesting increased vasodilation and blood flowin response to intra-arterial infusion (47) and exogenous administrationof ATP. These changes in blood flow likely lead to an increasedsubstrate pool for skeletal muscle by virtue of increased glucose and O₂uptake (42). The outcome is maintenance of energy status in the cellunder fatiguing contractions. (54, 56)

The physiological effects of ATP have led researchers to investigate theefficacy of oral supplementation of ATP (24). Jordan et al. (32)demonstrated that 225 mg per day of enteric-coated ATP supplementationfor 15 days resulted in increased total bench press lifting volume (i.e.sets·repetitions·load) as well as within-group set-one repetitions tofailure. More recently, Rathmacher et al. (52) found that 15 days of 400mg per day of ATP supplementation increased minimum peak torque in settwo of a knee extensor bout. Collectively the results discussed indicatethat ATP supplementation maintains performance and increases trainingvolume under high fatiguing conditions. However, greater fatigueincreases recovery demands between training sessions.

Current evidence suggests that HMB acts by speeding regenerativecapacity of skeletal muscle following high intensity or prolongedexercise (3). When training and/or diet are controlled, HMB can lowerindices of skeletal muscle damage and protein breakdown in adose-dependent fashion (50, 3, 2). Recently, HMB in a free acid form(HMB-FA) has been developed with improved bioavailability (18). Initialstudies have shown that this form of HMB supplementation results inapproximately double the plasma levels of HMB in about one-quarter thetime after administration when compared with the presently availableform, calcium HMB.

Further, HMB-FA given 30 minutes prior to an acute bout of high volumeresistance training was able to attenuate indices of muscle damage andimprove perceived recovery in resistance trained athletes (61). Moreoveracute ingestion of 2.4 grams of HMB-FA increases skeletal muscle proteinsynthesis and decreases protein breakdown by +70% and −56% respectively(58).

A need exists for a composition and methods to increase strength andpower and improve muscle mass. In addition, a need exists for acomposition that prevents or lessens the typical decay seen inperformance following an overreaching cycle. The present inventioncomprises a composition and methods of using a combination of ATP andHMB that results in these improvements.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a composition for usein increasing strength and power.

A further object of the present invention is to provide a compositionfor use in improving muscle mass.

Another object of the present invention is to provide methods ofadministering a composition for increasing strength and power.

An additional object of the present invention is to provide methods ofadministering a composition for improving muscle mass.

Another object of the present invention is to provide a composition foruse in preventing or lessening decay seen in performance following anoverreaching cycle.

These and other objects of the present invention will become apparent tothose skilled in the art upon reference to the following specification,drawings, and claims.

The present invention intends to overcome the difficulties encounteredheretofore. To that end, a composition comprising HMB and ATP isprovided. The composition is administered to an animal in need thereof.All methods comprise administering to the animal HMB and ATP.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schemata of phases of the training program listing variablesand the time points of measurement throughout the study.

FIG. 2 shows total strength, 1-RM Change from 8 10 12 weeks.

FIGS. 3a-c show changes in squat strength and bench press strength.

FIGS. 4a-b show the percent increase in vertical jump power and Wingatepeak power.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly and unexpectedly discovered that a combinationof HMB and ATP results in greater increases in strength, power andmuscle mass than use of either HMB or ATP alone. The present inventioncomprises a combination of HMB and ATP that has a synergistic effect andincreases strength and power. The present invention also comprises acombination of HMB and ATP that has the unexpected and surprisingresults of improving muscle mass. The present invention also comprises acombination of HMB and ATP that has the unexpected and surprising resultof preventing or lessening typical decay seen in performance followingan overreaching cycle. The combination of HMB and ATP results insignificant enhancements.

This combination can be used on all age groups seeking increases instrength and power, increases in muscle mass, and prevention orlessening of typical decay seen in performance following an overreachingcycle.

In view of the above, in one embodiment the present invention provides acomposition comprising HMB and ATP.

HMB

β-hydroxy-β-methylbutyric acid, or β-hydroxy-isovaleric acid, can berepresented in its free acid form as (CH₃)₂(OH)CCH₂COOH. The term “HMB”refers to the compound having the foregoing chemical formula, in bothits free acid and salt forms, and derivatives thereof. While any form ofHMB can be used within the context of the present invention, preferablyHMB is selected from the group comprising a free acid, a salt, an ester,and a lactone. HMB esters include methyl and ethyl esters. HMB lactonesinclude isovalaryl lactone. HMB salts include sodium salt, potassiumsalt, chromium salt, calcium salt, magnesium salt, alkali metal salts,and earth metal salts.

Methods for producing HMB and its derivatives are well-known in the art.For example, HMB can be synthesized by oxidation of diacetone alcohol.One suitable procedure is described by Coffman et al., J. Am. Chem. Soc.80: 2882-2887 (1958). As described therein, HMB is synthesized by analkaline sodium hypochlorite oxidation of diacetone alcohol. The productis recovered in free acid form, which can be converted to a salt. Forexample, HMB can be prepared as its calcium salt by a procedure similarto that of Coffman et al. (1958) in which the free acid of HMB isneutralized with calcium hydroxide and recovered by crystallization froman aqueous ethanol solution. The calcium salt of HMB is commerciallyavailable from Metabolic Technologies, Ames, Iowa.

Calcium β-hydroxy-β-methylbutyrate (HMB) Supplementation

More than 2 decades ago, the calcium salt of HMB was developed as anutritional supplement for humans. Numerous studies have shown thatCaHMB supplementation improves muscle mass and strength gains inconjunction with resistance-exercise training, and attenuates loss ofmuscle mass in conditions such as cancer and AIDS (1-5). Nissen andSharp performed a meta-analysis of supplements used in conjunction withresistance training and found that HMB was one of only two supplementsthat had clinical studies showing significant increases in strength andlean mass with resistance training (1). Studies have shown that 38 mg ofCaHMB per kg of body weight appears to be an efficacious dosage for anaverage person (6).

In addition to strength and muscle mass gains, CaHMB supplementationalso decreases indicators of muscle damage and protein degradation.Human studies have shown that muscle damage following intense exercise,measured by elevated plasma CPK (creatine phosphokinase), is reducedwith HMB supplementation. The protective effect of HMB has been shown tomanifest itself for at least three weeks with continued daily use (6-8)In vitro studies in isolated rat muscle show that HMB is a potentinhibitor of muscle proteolysis (9) especially during periods of stress.These findings have been confirmed in humans; for example, HMB inhibitsmuscle proteolysis in subjects engaging in resistance training (3).

The molecular mechanisms by which HMB decreases protein breakdown andincreases protein synthesis have been reported (10, 11). Eley et alconducted in vitro studies which have shown that HMB stimulates proteinsynthesis through mTOR phosphorylation (11, 12). Other studies haveshown HMB decreases proteolysis through attenuation of the induction ofthe ubiquitin-proteosome proteolytic pathway when muscle proteincatabolism is stimulated by proteolysis inducing factor (PIF),lipopolysaccharide (LPS), and angiotension II (10, 13, 14). Still otherstudies have demonstrated that HMB also attenuates the activation ofcaspases-3 and -8 proteases (15). Taken together these studies indicatethat HMB supplementation results in increased lean mass and theaccompanying strength gains through a combination of decreasedproteolysis and increased protein synthesis.

HMB Free Acid Form

In most instances, the HMB utilized in clinical studies and marketed asan ergogenic aid has been in the calcium salt form (3, 16). Recentadvances have allowed the HMB to be manufactured in a free acid form foruse as a nutritional supplement. Recently, a new free acid form of HMBwas developed, which was shown to be more rapidly absorbed than CaHMB,resulting in quicker and higher peak serum HMB levels and improved serumclearance to the tissues (18).

HMB free acid may therefore be a more efficacious method ofadministering HMB than the calcium salt form, particularly whenadministered directly preceding intense exercise. HMB free acidinitiated 30 min prior to an acute bout of exercise was more efficaciousin attenuating muscle damage and ameliorating inflammatory response thanCaHMB. One of ordinary skill in the art, however, will recognize thatthis current invention encompasses HMB in any form.

HMB in any form may be incorporated into the delivery and/oradministration form in a fashion so as to result in a typical dosagerange of about 0.5 grams HMB to about 30 grams HMB.

Adenosine-5′-triphosphate (ATP)

Supplementation with adenosine-5′-triphosphate (ATP) has been used toelevate extracellular ATP levels. Studies have failed to show consistentpositive effects of ATP to improve strength or power when combined withresistance-training exercise; however, small and transient increases insystemic ATP have been shown to increase blood flow in muscle tissue.

Oral administration of ATP is usually in the form ofAdenosine-5′-Triphospate Disodium. In the present invention,Adenosine-5′-Triphosphate Disodium or any form of ATP or adenosinesuitable for oral administration may be combined with any of the knowncoatings suitable for imparting enteric properties in granular form.

ATP may be incorporated into the delivery and/or administration form ina fashion so as to result in a typical dosage range of about 10 mg toabout 80 grams, though more or less may be desirable depending on theapplication and other ingredients.

The composition of HMB and ATP is administered to an animal in anysuitable manner. Acceptable forms include, but are not limited to,solids, such as tablets or capsules, and liquids, such as enteral orintravenous solutions. Also, the composition can be administeredutilizing any pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are well known in the art and examples of suchcarriers include various starches and saline solutions. In the preferredembodiment, the composition is administered in an edible form. Inaddition, an effective dosage range may be administered in divideddosages, such as two to three times per day.

ATP and HMB Combination

Any suitable dose of HMB can be used within the context of the presentinvention. Methods of calculating proper doses are well known in theart. The dosage amount of HMB can be expressed in terms of correspondingmole amount of Ca-HMB. The dosage range within which HMB may beadministered orally or intravenously is within the range from 0.01 to0.5 grams HMB (Ca-HMB) per kilogram of body weight per 24 hours. Foradults, assuming body weights of from about 100 to 200 lbs., the dosageamount orally or intravenously of HMB (Ca-HMB basis) can range from 0.5to 30 grams per subject per 24 hours.

ATP is present in the composition in any form. A range of ATP in thepresent invention includes ATP in the amount of around 10 milligrams toaround 80 grams.

When the composition is administered orally in an edible form, thecomposition is preferably in the form of a dietary supplement, foodstuffor pharmaceutical medium, more preferably in the form of a dietarysupplement or foodstuff. Any suitable dietary supplement or foodstuffcomprising the composition can be utilized within the context of thepresent invention. One of ordinary skill in the art will understand thatthe composition, regardless of the form (such as a dietary supplement,foodstuff or a pharmaceutical medium), may include amino acids,proteins, peptides, carbohydrates, fats, sugars, minerals and/or traceelements.

In order to prepare the composition as a dietary supplement orfoodstuff, the composition will normally be combined or mixed in such away that the composition is substantially uniformly distributed in thedietary supplement or foodstuff. Alternatively, the composition can bedissolved in a liquid, such as water.

The composition of the dietary supplement may be a powder, a gel, aliquid or may be tabulated or encapsulated.

Although any suitable pharmaceutical medium comprising the compositioncan be utilized within the context of the present invention, preferably,the composition is combined with a suitable pharmaceutical carrier, suchas dextrose or sucrose.

Furthermore, the composition of the pharmaceutical medium can beintravenously administered in any suitable manner. For administrationvia intravenous infusion, the composition is preferably in awater-soluble non-toxic form. Intravenous administration is particularlysuitable for hospitalized patients that are undergoing intravenous (IV)therapy. For example, the composition can be dissolved in an IV solution(e.g., a saline or glucose solution) being administered to the patient.Also, the composition can be added to nutritional IV solutions, whichmay include amino acids, peptides, proteins and/or lipids. The amountsof the composition to be administered intravenously can be similar tolevels used in oral administration. Intravenous infusion may be morecontrolled and accurate than oral administration.

Methods of calculating the frequency by which the composition isadministered are well-known in the art and any suitable frequency ofadministration can be used within the context of the present invention(e.g., one 6 g dose per day or two 3 g doses per day) and over anysuitable time period (e.g., a single dose can be administered over afive minute time period or over a one hour time period, or,alternatively, multiple doses can be administered over an extended timeperiod). The combination of HMB and ATP can be administered over anextended period of time, such as weeks, months or years.

It will be understood by one of ordinary skill in the art that HMB andATP do not have to be administered in the same composition to performthe claimed methods. Stated another way, separate capsules, pills,mixtures, etc. of ATP and of HMB may be administered to a subject tocarry out the claimed methods.

Any suitable dose of HMB can be used within the context of the presentinvention. Methods of calculating proper doses are well known in theart. Likewise, any suitable dose of ATP can be used within the contextof the present invention. Methods of calculating proper doses are wellknown in the art.

In general, an amount of HMB and ATP in the levels sufficient toincrease strength and power is described. Both HMB free acid alone andHMB free acid plus ATP supplementation increased strength and powergains greater than those observed with placebo supplementation (p<0.001,treatment*time). Surprisingly, post hoc analysis showed that HMB plusATP supplementation significantly further improved strength and powergains over those for HMB supplementation alone (p<0.05). The followingexperimental examples indicate that HMB does have a positive effect onstrength, power, and muscle mass and reduces muscle damage while aidingin recovery. Surprisingly, the combination of HMB plus ATP resulted ineven greater improvement in strength and power compared to HMB alone andthese effects are synergistic. Additionally, the HMB-ATP combinationalso demonstrated surprising and unexpected effects on muscle mass anddeclines in performance that are characteristic of overreaching.

EXPERIMENTAL EXAMPLES

The following examples will illustrate the invention in further detail.It will be readily understood that the composition of the presentinvention, as generally described and illustrated in the Examplesherein, could be synthesized in a variety of formulations and dosageforms. Thus, the following more detailed description of the presentlypreferred embodiments of the methods, formulations and compositions ofthe present invention are not intended to limit the scope of theinvention, as claimed, but it is merely representative of the presentlypreferred embodiments of the invention.

In the examples, overreaching is an increase in training volume and/orintensity of exercise resulting in performance decrements. Recovery fromthis condition often requires a few days to a week or more. Manystructured training programs utilize phases of overreaching to induce anadaptive response.

Lean body mass (LBM) and hypertrophy increases are used as indicators ofimproving muscle mass.

Study Design

The current study was a randomized, double-blind, placebo- anddiet-controlled experiment consisting of 12 weeks of periodizedresistance training. The training protocol was divided into 3 phases(Tables 1, 2, and 3). Phase 1 consisted of a non-linear periodizedresistance training program (3 times per week) modified from Kraemer etal. (36) (Table 1).

TABLE 1 Phase 1 of the training cycle (Daily Undulating Periodization).Monday Wednesday Friday Squat Squat Squat (5 sets) Barbell Bench PressBench Barbell Bench Press (5 sets) Deadlifts Deadlift Deadlifts PullUps/Dips (Superset) Pull Ups/Dumbbell Shoulder Press (Superset) BentOver Row Bent Over Row Dumbbell Shoulder Press Dumbbell Shoulder PressBarbell Curl/Triceps Barbell Curls/Triceps Extension (Superset)Extension (Superset) Repetition, Set Schema Repetition, Set SchemaRepetition, Set Schema 3 sets 5 sets 3 sets *(Squat and Bench = 5 sets)8-12 RM loads 5 maximal intended velocity 3-5 RM loads repetitions 60seconds timed rest 180 seconds timed rest 240 seconds timed rest

Phase 2 (Table 2) consisted of a two week overreaching cycle.

TABLE 2 Phase 2 of the training cycle (Overreaching). Monday TuesdayWednesday Thursday Friday Saturday Squat Leg Press Squat Leg Press Squat1 RM Wingate and Maximal Power Testing Barbell Bench Barbell BenchBarbell Bench Barbell Bench Bench Press 1 Press Press Press Press RMDeadlifts Military Press Deadlifts Military Press Deadlift 1 RM PullUps/Dips Supinated Pull Pull Ups/Dips Supinated Pull (Superset) Ups/Dips(Superset) Ups/Dips (Superset) (Superset) Bent Over Row Bent Over RowBent Over Row Bent Over Row Dumbbell Shoulder Hammer Curls/ DumbbellHammer Curls/ Press Close Grip Bench Shoulder Press Close Grip(Superset) Bench (Superset) Barbell Curl/ Barbell Curl/ TricepsExtension Triceps (Superset) Extension (Superset) Repetition, SetRepetition, Set Repetition, Set Repetition, Set Repetition, SchemaSchema Schema Schema Set Schema 3 sets 3 sets 3 sets 3 sets 3 MaximalAttempts (Highest Counted as 1RM) 8 RM loads 8 RM loads 12 RM loads 12RM loads 1 RM loads 60 seconds timed 60 seconds timed 60 seconds timed60 seconds 5 minutes rest rest rest timed rest timed rest 75% 1 RM 75% 1RM 65% 1 RM 65% 1 RM

Finally, phase 3 consisted of a tapering of the training volume forweeks 11 and 12 (Table 3).

TABLE 3 Phase 3 of the training cycle (Taper). Week 11 Week 12 MondayWednesday Friday Monday Wednesday Friday Squat Squat (3 sets) SquatSquat (3 sets) Squat Squat 1 RM Barbell Bench Barbell Bench BarbellBarbell Bench Barbell Bench Bench Press 1 RM Press Press Bench PressPress Press (3 sets) (3 sets) Deadlifts Deadlifts Deadlifts DeadliftsDeadlifts Deadlift 1 RM Pull Pull Ups/Dumbbell Ups/Dumbbell ShoulderPress Shoulder Press (Superset) (Superset) Bent Over Row Bent Over RowDumbbell Dumbbell Shoulder Press Shoulder Press Barbell Curls/ BarbellCurls/ Triceps Triceps Extension Extension (Superset) (Superset)Repetition, Repetition, Set Repetition, Repetition, Repetition, SetRepetition, Set Set Schema Schema Set Schema Set Schema Schema Schema 5sets 1 set *(Squat 5 sets 1 set *(Squat 5 sets 3 Maximal and Bench = 3and Bench = 3 Attempts (Highest sets) sets) Counted as 1RM) 5 maximal3-5 RM loads 5 maximal 3-5 RM loads 5 maximal 1 RM load intendedintended intended velocity velocity velocity repetitions repetitionsrepetitions 180 seconds 240 seconds 180 seconds 240 seconds 180 seconds5 minutes timed timed rest timed rest timed rest timed rest timed restrest 40-60% 1RM >90% 1 RM 40-60% >90% 1 RM 40-60% 1RM 1RM

Muscle mass, body composition, strength, power, resting plasmatestosterone, cortisol concentrations, and creatine kinase were examinedcollectively at the end of weeks 0, 4, 8, 9, 10, and 12 to assess thechronic effects of HMB-ATP; these were also assessed at the end of weeks9 and 10, corresponding to the mid- and endpoints of the phase 2overreaching cycle. An overview of the study design is summarized inFIG. 1.

Participants

Forty resistance-trained males aged 23.0±0.9 years with an averagesquat, bench press, and deadlift of 1.7±0.04, 1.3±0.04 and 2.0±0.05times their bodyweight were recruited for the study. Subjectcharacteristics are represented in Table 4. Participants could notparticipate if they were currently taking anti-inflammatory agents, anyother performance-enhancing supplement, or if they smoked. Eachparticipant signed an informed consent approved by the University ofTampa Institutional Review Board before participating in the study.

TABLE 4 Subject Descriptors. Treatments HMB-FA Placebo HMB-FA ATP plusATP N 10 11 11 8 Age, y 23.0 ± 1.2 21.3 ± 0.6 23.7 ± 0.9 21.4 ± 0.3 BodyWeight, 87.4 ± 4.3 83.1 ± 2.8 85.7 ± 1.7 81.9 ± 2.1 kg Height, cm 180.6± 2.3  179.0 ± 2.1  179.0 ± 1.0  177.2 ± 1.3  Body Mass 26.6 ± 0.7 25.9± 0.7 26.7 ± 0.4 26.1 ± 0.6 IndexMuscle Strength, Power, Body Composition and Skeletal Muscle HypertrophyTesting

After familiarization with procedures, muscle strength was assessed via1RM testing of the back squat, bench press, and deadlift. Each lift wasperformed as described by the International Powerlifting Federationrules (44). Body composition (lean body mass, fat mass, and total mass)was determined by dual x-ray absorptiometry (DXA; Lunar Prodigy enCORE2008, Madison, Wis., U.S.A.). Skeletal muscle hypertrophy was determinedvia the combined changes in ultrasonography-determined muscle thicknessof the vastus lateralis (VL) and vastus intermedius (VI) muscles. Theintraclass correlation coefficient (ICC) for the test-retest of musclethickness measurements was r=0.97.

Muscle power was assessed during maximal cycling (Wingate Test) andjumping movements. During the cycling test, volunteers were instructedto cycle against a predetermined resistance (7.5% of body weight) asfast as possible for 10 seconds (36). The saddle height was adjusted tothe individual's height to produce a 5-10° knee flexion while the footwas in the low position of the central void. A standardized verbalstimulus was provided to each participant. Power output was recorded inreal time during the 10-second sprint test, by a computer connected tothe standard cycle ergometer (Monark model 894e, Vansbro, Sweden). Peakpower (PP) was recorded using Monark Anaerobic Wingate Software, Version1.0 (Monark, Vansbro, Sweden). The ICC of muscle peak power was 0.96.

Measurements of PP were also taken during a vertical jump (VJ) testperformed on a multi-component AMTI force platform (Advanced MechanicalTechnology, Inc., Watertown, Mass.), interfaced with a personal computerat a sampling rate of 1000 Hz (51). Data acquisition software (LabVIEW,version 7.1; National Instruments Corporation, Austin, Tex.) was used tocalculate PP. Peak power was calculated as the peak combination ofground reaction force and peak velocity during the accelerated launch onthe platform. The ICC of VJ power was 0.97.

Supplementation, Diet Control, and Exercise Protocol

Prior to the study, participants were randomly assigned to receiveeither 3 g per day of HMB Free Acid (HMB) (combined with food-gradeorange flavors and sweeteners), 400 mg per day of ATP (PEAK ATP®; TSI,Inc.), a combination of both 3 g of HMB and 400 mg per day of ATP, or aplacebo (food-grade orange flavors and sweeteners) divided equally intothree servings daily with the first serving given 30 minutes prior toexercise and the remaining two servings daily with the mid-day andevening meals. On the non-training days participants were instructed toconsume one serving with each of three separate meals. Thesupplementation was continued daily throughout the training and testingprotocols. Each serving was formulated with 1 gram of HMB free acid toaccount for fill and emptying variation and achieve a minimum effectivedosage of 0.800 grams. This dosage would be equivalent to a 1 gramCa-HMB dosage.

The participants must not have taken any nutritional supplements for atleast three months prior to the start of data collection. Two weeksprior to and throughout the study, participants were placed on a dietconsisting of 25% protein, 50% carbohydrates, and 25% fat by aregistered dietician who specialized in sports nutrition. Theparticipants met as a group with the dietitian, and they were givenindividual meal plans at the beginning of the study. Diet counseling wascontinued on an individual basis throughout the study.

All participants performed a high volume resistance training protocolduring the 12-week study. The phases of the study and measurements takenare shown in FIG. 1, and the exercise protocols for each phase of thestudy are shown in Tables 1 to 3. The training was divided into 3phases, with Phase 1 consisting of daily undulating periodization (weeks1 to 8), Phase 2 consisting of the overreaching cycle (weeks 9 and 10),and Phase 3 consisting of the taper cycle (weeks 11 and 12).

Resting Blood Draws

All blood draws throughout the study were obtained via venipunctureafter a 12-hour fast by a trained phlebotomist. Whole blood wascollected and transferred into appropriate tubes for obtaining serum andplasma and centrifuged at 1,500 g for 15 min at 4° C. Resulting serumand plasma were then aliquoted and stored at −80° C. until subsequentanalyses.

Biochemical Analysis

Samples were thawed one time and analyzed in duplicate for each analyte.All blood draws were scheduled at the same time of day to negateconfounding influences of diurnal hormonal variations. Serum total andfree testosterone, cortisol, and C-reactive protein (CRP) were assayedvia ELISA kits obtained from Diagnostic Systems Laboratories (Webster,Tex.). All hormones were measured in the same assay on the same day toavoid compounded interassay variance. Intra-assay variance was less than3% for all analytes. Serum creatine kinase (CK) was measured usingcolorimetric procedures at 340 nm (Diagnostics Chemicals, Oxford,Conn.). Twenty-four hour urine collections were made and 3-methylhistinewas determined by previously described methods (Rathmacher et al 1992and Wilson et al, 2013).

Perceived Recovery Status Scale

Perceived Recovery Status (PRS) scale was measured at weeks 0, 4, 8, 9,10, and 12 to assess subjective recovery during the training phases. ThePRS scale consists of values between 0-10, with 0-2 being very poorlyrecovered and with anticipated declines in performance, 4-6 being low tomoderately recovered and expected similar performance, and 8-10representing high perceived recovery with expected increases inperformance.

Statistics

A one-way ANOVA model was used to analyze the baseline characteristicdata using the Proc GLM procedure in SAS (Version 9.1, SAS Institute,Cary, N.C.)¹ (SAS Institute, Inc. (1985) SAS User's Guide: Statistics,5th ed. Cary, N.C.: SAS Institute, Inc.). The main effect of treatment(Trt) was included in the model. Muscle strength and power, bodycomposition, muscle damage, hormonal status, and perceived recoveryscore (PRS) changes over the 12-week study were analyzed with a 2×2factorial, repeated measures ANOVA using the Proc Mixed procedure inSAS. The initial value, week 0, was used as a covariate with the maineffects of HMB, ATP, and Time, and the interactions of HMB*time,ATP*time, and HMB*ATP*time in the model. The overreaching cycle of thestudy was also assessed by using the 2×2 factorial, repeated measuresANOVA with the Proc Mixed procedure in SAS; however, the value measuredat the week 8 time point was used as a covariate with the main effectsof HMB, ATP, and Time, and the interactions of HMB*time, ATP*time andHMB*ATP*time. The Least Squares Means procedure was then used to comparetreatment means at each time point (post-hoc t-test). Statisticalsignificance was determined at p≤0.05 and trends were determined betweenp>0.05 and p≤0.10.

Results

Participant Characteristics

There were no differences in age (Placebo=23.0±1.2, ATP=23.7±0.9 yrs.,HMB=22.3±0.6, HMB-ATP=22.4±0.5), height (Placebo=180.6±2.3,ATP=179.0±1.0 cm, HMB=179.3±2.1, HMB-ATP=180.0±1.4), or body mass(Placebo=87.4±4.3, ATP=85.7±1.7, HMB=83.1±1.6, HMB-ATP=84.6±2.2) amongthe treatments at the start of the study.

Muscle Strength and Power

At weeks 0, 4, 8, 9, 10, and 12 during the study muscle strength (1-RMof squat, bench press, and deadlift) and muscle power (vertical jump andWingate Peak Power, PP) were measured; both muscle strength and powerincreased over the 12-week study (Time, p<0.001). Supplementation withHMB, ATP and the HMB-ATP combination increased total strength gains by77.1±5.6, 55.3±6.0, and 96.0±8.2 kg, respectively, compared with theplacebo-supplemented participants who gained 22.4±7.1 kg in totalstrength over the 12-week study (t-test, p<0.05). FIGS. 2 and 3 a-c showthe synergistic effect of HMB and ATP on strength. FIG. 2 shows totalstrength changes from weeks 8-12. FIGS. 3a-c show individual indicatorsof the synergistic combination, including squat strength and bench pressstrength from weeks 4-8 and 4-12.

During the overreaching cycle in weeks 9 and 10, total strength declinedin the placebo-supplemented participants by −4.5±0.9% from weeks 8 to10. Total strength decreased to a lesser extent in the ATP-supplementedsubjects by −2.±0.5% from week 8 to week 10 and at week 10 theATP-supplemented participants had increased total strength compared withthe placebo-supplemented participants (t-test, p<0.05). During theoverreaching cycle, HMB-supplementation attenuated the decrease in totalstrength (−0.5±1.2%, t-test, p<0.05 vs. placebo) and the HMB-ATPsupplemented subjects unexpectedly continued to increase in strength(1.2±0.7%, t-test, p<0.05 versus placebo).

Muscular power was assessed using both the vertical jump and Wingate PPtests and results are shown in FIGS. 4a and 4b , respectively. Both ofthese measures of power were significantly increased during the studywith HMB (HMB*time, p<0.001 for both) and with ATP supplementation(ATP*time, p<0.001 and p<0.04 for vertical jump power and Wingate PP,respectively, FIGS. 4A and 4B). Over the 12-weeks of training verticaljump power increased 614±52, 991±51, 796±75, and 1076±40 watts inplacebo, HMB, ATP, and HMB-ATP supplemented groups, respectively (t-testp<0.05). The percentage increases in vertical jump power weresynergistic with HMB and ATP supplemented in combination (HMB*ATP*time,p<0.004, FIG. 4a ). Vertical jump power during the overreaching cycledecreased more in the placebo group, 5.0±0.4%, compared with the smallerdecreases in vertical jump power for the HMB, ATP, and HMB-ATPsupplemented groups, 1.4±0.4, 2.2±0.4, and 2.2±0.5%, respectively, overweeks 9 and 10 (t-test, p<0.05, FIG. 4A). During the 2-week overreachingcycle, Wingate PP decreased by 4.7±1.5, 0.3±0.9, 2.9±0.7, and 2.0±0.9%in the placebo, HMB, ATP, and HMB-ATP supplemented groups, respectively(FIG. 4B). After the first week of the increased training, HMB, ATP, andHMB-ATP supplementation resulted in the participants maintaining greaterWingate PP power than the placebo supplemented group with gains in powerof 10.2±1.6, 9.0±1.6, and 14.5±1.2% from baseline, respectively (t-test,p<0.05). However, after the second week of the overreaching cycle onlythe HMB-ATP supplemented group had maintained significantly greaterWingate PP than the placebo-supplemented group, 1022±21 and 940±66watts, respectively (t-test, p<0.05, FIG. 4B).

Body Composition and Muscle Hypertrophy

Resistance training resulted in increased lean body mass (LBM) andquadriceps thickness (Time, p<0.001) whereas, fat percentage wasdecreased with the training, (Time, p<0.001) at weeks 0, 4, 8, and 12.Supplementation with HMB increased body weight, LBM, and quadricepsthickness and decreased body fat (HMB*time, p<0.03, p<0.001, p<0.001,and p<0.001, respectively) whereas ATP supplementation increased LBM andquadriceps thickness (ATP*time, p<0.01 and 0.04, respectively). Leanbody mass was increased in an additive manner by 2.1±0.5, 7.4±0.4,4.0±0.4, and 8.5±0.8 kg in placebo, HMB, ATP, and HMB-ATP supplementedparticipants, respectively (t-test, p<0.05, Table 5), and fat percentagedecreased by 7.0±0.6 and 8.5±0.9% in HMB and HMB-ATP supplementedparticipants, respectively (t-test p<0.05). Only the HMB supplementationwas shown to have a significant effect on fat percentage (HMB*timep<0.001). There was no main effect of ATP*time during the study on bodyweight; however, the ATP alone supplemented group did have a greaterbody weight by week 12 of the study than the placebo-supplemented group(t-test, p<0.05). The 12-week increases in quadriceps thicknesses were2.5±0.6, 7.1±1.2, 4.9±1.0, and 7.8±0.4 mm in placebo, HMB, ATP, andHMB-ATP supplemented participants, respectively, and HMB, ATP, andHMB-ATP supplementation resulted in a greater 12 week quadricepsthickness compared with placebo supplementation (t-test, p<0.05, Table5).

TABLE 5 Effect of Beta-hydroxy-Beta-methylbutyrate free acid (HMB-FA)and adenosine-5′-triphosphate (ATP) supplementation on weight, lean bodymass (LBM), percent body fat, and quadriceps muscle thickness insubjects performing a 12 week weight training regimen.^(a) Week of StudyMain Effects^(b) 0 4 8 12 HMB-FA*Time ATP*Time HMB-FA*ATP*Time Weight,kg Placebo 87.4 ± 4.3 88.3 ± 4.6 88.7 ± 4.8 87.7 ± 4.7  0.03 0.63 0.42HMB-FA 83.1 ± 2.8 83.9 ± 2.8 84.8 ± 2.9 85.0 ± 3.0^(#) ATP 85.7 ± 1.786.9 ± 2.0 87.0 ± 2.0 87.0 ± 2.1^(#) HMB Plus ATP 81.9 ± 2.1 82.9 ± 1.983.4 ± 1.9 83.6 ± 1.9^(#) DXA LBM, kg Placebo 68.5 ± 2.6 70.0 ± 2.3 71.2± 2.4 70.5 ± 2.4  0.001 0.01 0.80 HMB-FA 66.2 ± 2.6  69.2 ± 2.7^(#) 71.3 ± 2.7^(#) 73.5 ± 2.7^(#) ATP 67.7 ± 2.0 70.1 ± 1.9 71.4 ± 2.0 71.7± 1.9^(#) HMB Plus ATP 67.0 ± 1.2  70.5 ± 1.3^(#)  72.5 ± 1.6^(#) 75.4 ±1.5^(#) DXA Fat, % Placebo 21.0 ± 1.1 19.8 ± 1.6 18.6 ± 1.9 18.6 ± 1.7 0.001 0.28 0.99 HMB-FA 20.4 ± 1.4 17.6 ± 1.4  15.9 ± 1.5^(#) 13.5 ±1.5^(#) ATP 19.5 ± 1.8 18.1 ± 1.8 16.6 ± 1.6 16.0 ± 1.5  HMB Plus ATP18.0 ± 1.9  14.7 ± 2.2^(#)  12.7 ± 2.5^(#)  9.5 ± 2.2^(#) Quad, mmPlacebo 50.2 ± 2.1 52.2 ± 2.3 52.6 ± 2.4 52.7 ± 2.4  0.001 0.04 0.45HMB-FA 50.7 ± 1.5 53.6 ± 1.4  56.0 ± 1.4^(#) 57.8 ± 1.6^(#) ATP 50.9 ±0.9 53.4 ± 1.3 54.8 ± 1.7 55.8 ± 1.8^(#) HMB Plus ATP 50.5 ± 1.2 53.9 ±1.2  57.0 ± 1.2^(#) 58.3 ± 1.1^(#)Muscle Damage, Hormonal Status and Performance Recovery Scale

Muscle damage was assessed by blood CK, which was increased by training,particularly after the changes in training volume at the initiation ofthe study and at weeks 9 and 10 during the overreaching cycle (Table 6;Time, p<0.001). The initial training resulted in a 342±64% increase andthe two-week overreaching cycle resulted in a 159±55% increase in CKlevels in the placebo-supplemented group. Supplementation with HMBsignificantly attenuated the increase in CK at both the initiation oftraining (weeks 0 to 1) and during the overreaching cycle (weeks 9 and10) (HMB*time, p<0.001). Supplementation with ATP alone did notattenuate the increases in CK compared with the placebo supplementation;however, HMB-ATP supplementation resulted in a significant attenuationin CK increase compared with placebo at weeks 1, 4, 9, and 10 that wassimilar to the effect of HMB supplementation alone (t-test, p<0.05).

The rate of muscle protein degradation was evaluated by measuringurinary 3-MH:Cr ratio (Table 6).

TABLE 6 Effect of Beta-hydroxy-Beta-methylbutyrate free acid (HMB-FA)and adenosine-5′-triphosphate (ATP) supplementation blood creatinekinase (CK), C-reactive protein (CRP), cortisol, free and totaltestosterone, lactate dehydrogenase (LDH) and perceived recovery score(PRS) in subjects performing a 12 week weight training regimen ^(a) Weekof Study 0 1 4 8 9 10 CK, IU/L Placebo 141 ± 12 582 ± 77 373 ± 13 246 ±29 484 ± 52  528 ± 72 HMB-FA 158 ± 16  322 ± 35^(#)  280 ± 22^(#) 255 ±28 288 ± 18^(# )  250 ± 14^(#) ATP 145 ± 8  500 ± 71 324 ± 14 234 ± 32426 ± 44  449 ± 62 HMB/ATP 162 ± 31  310 ± 42^(#)  232 ± 30^(#) 212 ± 22262 ± 23^(# )  269 ± 31^(#) 24 h 3MH:Cr, μmol:mg Placebo  0.127 ± 0.007 0.130 ± 0.003  0.123 ± 0.004 0.134 ± 0.005  0.152 ± 0.005 HMB-FA  0.127± 0.004  0.122 ± 0.002  0.124 ± 0.008 0.120 ± 0.003  0.141 ± 0.004 ATP 0.136 ± 0.008  0.127 ± 0.007   0.143 ± 0.007^(#) 0.143 ± 0.008   0.131± 0.012^(#) HMB/ATP  0.121 ± 0.007   0.153 ± 0.009^(#)  0.131 ± 0.0080.131 ± 0.009  0.142 ± 0.005 CRP, mg/L Placebo  1.9 ± 0.7  1.1 ± 0.1 1.3 ± 0.3  2.0 ± 0.7 1.6 ± 0.7  1.2 ± 0.2 HMB-FA  1.0 ± 0.1  1.3 ± 0.3 1.0 ± 0.1  0.9 ± 0.01 1.0 ± 0.1   1.8 ± 0.8^(#) ATP  1.4 ± 0.4  1.1 ±0.1  1.2 ± 0.2  1.9 ± 0.6 1.7 ± 0.6  1.1 ± 0.1 HMB/ATP  1.2 ± 0.2  1.6 ±0.6  1.1 ± 0.2  1.1 ± 0.2  0.9 ± 0.06  1.1 ± 0.1 Cortisol, μg/dL Placebo19.7 ± 1.1 20.8 ± 1.3 19.0 ± 1.2 19.2 ± 0.4 22.0 ± 0.4  23.6 ± 0.3HMB-FA 21.5 ± 1.4 20.3 ± 1.2 20.9 ± 1.0 18.8 ± 1.5 19.6 ± 1.1^(# )  18.6± 1.2^(#) ATP 20.9 ± 1.2 20.5 ± 1.3 18.4 ± 1.4 19.0 ± 0.4 21.5 ± 0.4 22.6 ± 0.2 HMB/ATP 20.4 ± 1.2  18.2 ± 2.2^(#) 17.7 ± 1.6 17.2 ± 1.5 19.1± 1.0^(# )  17.8 ± 1.9^(#) Free Testosterone, ng/dL Placebo 103 ± 13 112± 10 119 ± 6  111 ± 9  98 ± 6  100 ± 9  HMB-FA 109 ± 10 104 ± 8  116 ±11 115 ± 9  118 ± 8  116 ± 8  ATP 112 ± 13 114 ± 9  118 ± 6  117 ± 11108 ± 7  110 ± 10 HMB/ATP 90 ± 5 98 ± 5 116 ± 10 103 ± 14 102 ± 11  115± 12 Total Testosterone, ng/dL Placebo 591 ± 73 620 ± 58 625 ± 55 585 ±58 551 ± 46  536 ± 88 HMB-FA 708 ± 35 708 ± 48 730 ± 63 652 ± 35 752 ±61^(# ) 701 ± 34 ATP 660 ± 67 645 ± 54 695 ± 60 645 ± 60 621 ± 49  592 ±84 HMB/ATP 568 ± 39 583 ± 34 636 ± 49 533 ± 51 581 ± 71  617 ± 37PRS^(c) Placebo  9.1 ± 0.3  4.7 ± 0.4  7.0 ± 0.3  7.6 ± 0.2 4.8 ± 0.3 4.4 ± 0.3 HMB-FA  9.1 ± 0.3   6.3 ± 0.3^(#)  7.6 ± 0.3   8.5 ± 0.3^(#) 8.0 ± 0.2^(#)   7.7 ± 0.2^(#) ATP  9.6 ± 0.2  4.9 ± 0.4  7.5 ± 0.3  8.2± 0.3 5.5 ± 0.4   5.5 ± 0.4^(#) HMB/ATP  9.6 ± 0.2   6.6 ± 0.3^(#)   8.4± 0.2^(#)  8.5 ± 0.3  7.6 ± 0.2^(#)   7.4 ± 0.2^(#) Week of Study MainEffects^(b) 12 HMB-FA*Time ATP*Time HMB-FA*ATP*Time CK, IU/L Placebo 187± 21 0.001 0.89 0.85 HMB-FA 147 ± 15 ATP 160 ± 20 HMB/ATP 169 ± 15 24 h3MH:Cr, μmol:mg Placebo 0.05 0.009 0.005 HMB-FA ATP HMB/ATP CRP, mg/LPlacebo  1.6 ± 0.4 0.08 0.95 0.92 HMB-FA  1.1 ± 0.1 ATP  1.2 ± 0.2HMB/ATP  1.0 ± 0.1 Cortisol, μg/dL Placebo 20.3 ± 0.6 0.001 0.78 0.77HMB-FA  17.4 ± 1.2^(#) ATP 19.7 ± 0.6 HMB/ATP  16.8 ± 1.4^(#) FreeTestosterone, ng/dL Placebo 113 ± 12 0.21 0.96 0.76 HMB-FA 127 ± 8  ATP125 ± 13 HMB/ATP 118 ± 9  Total Testosterone, ng/dL Placebo 605 ± 720.18 0.93 0.93 HMB-FA 728 ± 39 ATP 673 ± 69 HMB/ATP 655 ± 27 PRS^(c)Placebo  7.6 ± 0.2 0.001 0.79 0.06 HMB-FA   9.5 ± 0.2^(#) ATP   8.6 ±0.4^(#) HMB/ATP   9.6 ± 0.2^(#) ^(a)Mean ± SEM for n = 10 placebo, n =11 HMB (3 g HMB free acid/d in three 1 g doses daily), n = 11 ATP (one400 mg dose of ATP in the morning), and n = 8 for HMB plus ATP (3 g HMBfree acid/d in three 1 g doses daily and one 400 mg dose of ATP in themorning) supplemented subjects. ^(b)Probability of treatment by timedifference between the treatments over the 12-week study. The mixedmodel 2 × 2 Factorial Repeat ANOVA (SAS ®) was used, with the value forweek 0 used as a covariate. ^(c)Percieved recovery score is rated on theparticipants feeling of recovery from the last workout on a scale of0-10. ^(#)Significantly different than corresponding placebo, t-test (p< 0.005).

C-Reactive protein levels were not significantly affected by any of thetreatments during the study. A trend was observed for an HMB effect(HMB*time, p<0.08) and HMB supplementation resulted in a greater meanCRP value at week 10 than did placebo supplementation (t-test, p<0.05).Supplementation with ATP did not affect cortisol levels, while HMBsupplementation decreased cortisol levels during the study (HMB*time,p<0.001, Table 6). Supplementation with HMB alone resulted in decreasedcortisol levels at weeks 9, 10, and 12 during the overreaching and tapercycles (t-test, p<0.05) and supplementation with HMB-ATP resulted indecreased cortisol levels after both the initiation of training, week 1,and the overreaching and taper cycles, weeks 9, 10, and 12 (t-testp<0.05). There were no main effect differences of either HMB or ATP oneither free or total testosterone.

Muscle recovery and readiness to train in the next training session weremeasured by perceived recovery score (PRS, Table 6). Supplementationwith HMB and HMB-ATP resulted in improved PRS over the 12-week study(HMB*time, p<0.001). While no main effect of ATP supplementation wasobserved, ATP-supplemented participants had improved PRS scores afterthe overreaching cycle at weeks 10 and 12 compared withplacebo-supplemented participants (t-test, p<0.05). At week 4, theHMB/ATP-supplemented group was the only group with a significantlyimproved PRS compared with the placebo-supplementation (t-test, p<0.05).A trend for an HMB and ATP interaction, indicating a synergistic effectof the combined supplementation on PRS, was also observed (HMB*ATP*time,p<0.06).

The experimental examples demonstrate that HMB-ATP supplementationresults in increased strength and power adaptations compared to just HMBor ATP supplementation alone, and this increase is synergistic.

Further, the results indicated greater increases in LBM and musclethickness in the HMB-ATP, HMB, and ATP groups as compared to the placeboand the administration of HMB-ATP has greater effects on musclehypertrophy and lean body mass compared to just HMB or ATPsupplementation alone.

The administration of HMB-ATP results in increases in LBM, musclehypertrophy, strength, and power. These increases are, in the instancesof strength and power, synergistic, and in the instances of lean bodymass and muscle hypertrophy, additive. Moreover, when faced with greatertraining frequencies, as demonstrated with the overreaching cycle oftraining, HMB-ATP prevents typical declines in performance that arecharacteristic of overreaching. All of these results were unexpected andsurprising.

The foregoing description and drawings comprise illustrative embodimentsof the present inventions. The foregoing embodiments and the methodsdescribed herein may vary based on the ability, experience, andpreference of those skilled in the art. Merely listing the steps of themethod in a certain order does not constitute any limitation on theorder of the steps of the method. The foregoing description and drawingsmerely explain and illustrate the invention, and the invention is notlimited thereto, except insofar as the claims are so limited. Thoseskilled in the art who have the disclosure before them will be able tomake modifications and variations therein without departing from thescope of the invention. The terms subject and animal are usedinterchangeably throughout this application and are in no way limited toone term or the other.

LITERATURE CITED

-   1. Nissen, S. L. & Sharp, R. L. Effect of dietary supplements on    lean mass and strength gains with resistance exercise: a    meta-analysis. J Appl. Physiol 94: 651-659, 2003.-   2. Panton, L. B., Rathmacher, J. A., Baier, S. & Nissen, S.    Nutritional supplementation of the leucine metabolite b-hydroxy    b-methylbutyrate (HMB) during resistance training. Nutr. 16(9):    734-739, 2000.-   3. Nissen, S., Sharp, R., Ray, M., Rathmacher, J. A., Rice, J.,    Fuller, J. C., Jr., Connelly, A. S. & Abumrad, N. N. Effect of the    leucine metabolite b-hydroxy b-methylbutyrate on muscle metabolism    during resistance-exercise training. J. Appl. Physiol. 81(5):    2095-2104, 1996.-   4. Eubanks May, P., Barber, A., Hourihane, A., D'Olimpio, J. T. &    Abumrad, N. N. Reversal of cancer-related wasting using oral    supplementation with a combination of b-hydroxy-b-methylbutyrate,    arginine, and glutamine. Am. J. Surg. 183: 471-479, 2002.-   5. Clark, R. H., Feleke, G., Din, M., Yasmin, T., Singh, G.,    Khan, F. & Rathmacher, J. A. Nutritional treatment for acquired    immunodeficiency virus-associated wasting using    b-hydroxy-b-methylbutyrate, glutamine and arginine: A randomized,    double-blind, placebo-controlled study. JPEN J Parenter Enteral Nutr    24(3): 133-139, 2000.-   6. Gallagher, P. M., Carrithers, J. A., Godard, M. P.,    Schulze, K. E. & Trappe, S. W. b-Hydroxy-b-methylbutyrate ingestion,    Part I: Effects on strength and fat free mass. Med Sci Sports Exerc    32(12): 2109-2115, 2000.-   7. Jówko, E., Ostaszewski, P., Jank, M., Sacharuk, J., Zieniewicz,    A., Wilczak, J. & Nissen, S. Creatine and b-hydroxy-b-methylbutyrate    (HMB) additively increases lean body mass and muscle strength during    a weight training program. Nutr. 17: 558-566, 2001.-   8. Knitter, A. E., Panton, L., Rathmacher, J. A., Petersen, A. &    Sharp, R. Effects of b-hydroxy-b-methylbutyrate on muscle damage    following a prolonged run. J. Appl. Physiol. 89(4): 1340-1344, 2000.-   9. Ostaszewski, P., Kostiuk, S., Balasinska, B., Jank, M., Papet, I.    & Glomot, F. The leucine metabolite 3-hydroxy-3-methylbutyrate (HMB)    modifies protein turnover in muscles of the laboratory rats and    domestic chicken in vitro. J. Anim. Physiol. Anim. Nutr. (Swiss) 84:    1-8, 2000.-   10. Russell, S. T. & Tisdale, M. J. Mechanism of attenuation by    beta-hydroxy-beta-methylbutyrate of muscle protein degradation    induced by lipopolysaccharide. Mol. Cell Biochem. 330 (1-2):    171-179, 2009.-   11. Eley, H. L., Russell, S. T. & Tisdale, M. J. Attenuation of    depression of muscle protein synthesis induced by    lipopolysaccharide, tumor necrosis factor and angiotensin II by    b-hydroxy-b-methylbutyrate. Am. J. Physiol Endocrinol. Metab 295:    E1409-E1416, 2008.-   12. Eley, H. L., Russell, S. T., Baxter, J. H., Mukerji, P. &    Tisdale, M. J. Signaling pathways initiated by    b-hydroxy-b-methylbutyrate to attenuate the depression of protein    synthesis in skeletal muscle in response to cachectic stimuli.    Am. J. Physiol Endocrinol. Metab 293: E923-E931, 2007.-   13. Smith, H. J., Wyke, S. M. & Tisdale, M. J. Mechanism of the    attenuation of proteolysis-inducing factor stimulated protein    degradation in muscle by beta-hydroxy-beta-methylbutyrate. Cancer    Res. 64: 8731-8735, 2004.-   14. Smith, H. J., Mukerji, P. & Tisdale, M. J. Attenuation of    proteasome-induced proteolysis in skeletal muscle by    b-hydroxy-b-methylbutyrate in cancer-induced muscle loss. Cancer    Res. 65: 277-283, 2005.-   15. Eley, H. L., Russell, S. T. & Tisdale, M. J. Mechanism of    Attenuation of Muscle Protein Degradation Induced by Tumor Necrosis    Factor Alpha and Angiotensin II by beta-Hydroxy-beta-methylbutyrate.    Am. J. Physiol Endocrinol. Metab 295: E1417-E1426, 2008.-   16. Fuller, J. C., Jr., Baier, S., Flakoll, P. J., Nissen, S. L.,    Abumrad, N. N. & Rathmacher, J. A. Vitamin D status affects strength    gains in older adults supplemented with a combination of    b-hydroxy-b-methylbutyrate, arginine and lysine: A cohort study.    JPEN 35: 757-762, 2011.-   17. Sousa, M. F., Abumrad, N. N., Martins, C., Nissen, S. &    Riella, M. C. Calcium b-hydroxy-b-methylbutyrate. Potential role as    a phosphate binder in uremia: In vitro study. Nephron 72: 391-394,    1996.-   18. Fuller, J. C., Jr., Sharp, R. L., Angus, H. F., Baier, S. M. &    Rathmacher, J. A. Free acid gel form of    beta-hydroxy-beta-methylbutyrate (HMB) improves HMB clearance from    plasma in human subjects compared with the calcium HMB salt. Br. J    Nutr. 105: 367-372, 2011.-   19. Kushmerick, M. J. & Conley, K. E. Energetics of muscle    contraction: the whole is less than the sum of its parts. Biochem.    Soc. Trans. 30: 227-231, 2002.-   20. Burnstock, G., Knight, G. E. & Greig, A. V. Purinergic signaling    in healthy and diseased skin. J Invest Dermatol. 132: 526-546, 2012.-   21. Agteresch, H. J., Dagnelie, P. C., van den Berg, J. W. &    Wilson, J. H. Adenosine triphosphate: established and potential    clinical applications. Drugs 58: 211-232, 1999.-   22. Sawynok, J. & Sweeney, M. I. The role of purines in nociception.    Neuroscience 32: 557-569, 1989.-   23. Yajima, H., Sato, J., Giron, R., Nakamura, R. & Mizumura, K.    Inhibitory, facilitatory, and excitatory effects of ATP and    purinergic receptor agonists on the activity of rat cutaneous    nociceptors in vitro. Neurosci. Res. 51: 405-416, 2005.-   24. Khakh, B. S. & Henderson, G. ATP receptor-mediated enhancement    of fast excitatory neurotransmitter release in the brain. Mol.    Pharmacol. 54: 372-378, 1998.-   25. Ellis, C. G., Milkovich, S. & Goldman, D. What is the Efficiency    of ATP Signaling from Erythrocytes to Regulate Distribution of O(2)    Supply within the Microvasculature? Microcirculation, 2012.-   26. Gergs, U., Boknik, P., Schmitz, W., Simm, A., Silber, R. E. &    Neumann, J. A positive inotropic effect of adenosine in cardiac    preparations of right atria from diseased human hearts. Naunyn    Schmiedebergs Arch. Pharmacol. 379: 533-540, 2009.-   27. Gergs, U., Boknik, P., Schmitz, W., Simm, A., Silber, R. E. &    Neumann, J. A positive inotropic effect of ATP in the human cardiac    atrium. Am. J Physiol Heart Circ. Physiol 294: H1716-H1723, 2008.-   28. Kichenin, K., Decollogne, S., Angignard, J. & Seman, M.    Cardiovascular and pulmonary response to oral administration of ATP    in rabbits. J Appl. Physiol 88: 1962-1968, 2000.-   29. Heinonen, I., Kemppainen, J., Kaskinoro, K., Peltonen, J. E.,    Sipila, H. T., Nuutila, P., Knuuti, J., Boushel, R. &    Kalliokoski, K. K. Effects of adenosine, exercise, and moderate    acute hypoxia on energy substrate utilization of human skeletal    muscle. Am. J. Physiol Regul. Integr. Comp Physiol 302: R385-R390,    2012.-   30. Yegutkin, G. G. Nucleotide- and nucleoside-converting    ectoenzymes: Important modulators of purinergic signalling cascade.    Biochim. Biophys. Acta 1783: 673-694, 2008.-   31. Nyberg, M., Mortensen, S. P., Thaning, P., Saltin, B. &    Hellsten, Y. Interstitial and plasma adenosine stimulate nitric    oxide and prostacyclin formation in human skeletal muscle.    Hypertension 56: 1102-1108, 2010.-   32. Jordan, A. N., Jurca, R., Abraham, E. H., Salikhova, A.,    Mann, J. K., Morss, G. M., Church, T. S., Lucia, A. & Earnest, C. P.    Effects of oral ATP supplementation on anaerobic power and muscular    strength. Med. Sci. Sports Exerc. 36: 983-990, 2004.-   33. Arts, I. C., Coolen, E. J., Bours, M. J., Huyghebaert, N., Cohen    Stuart, M. A., Bast, A. & Dagnelie, P. C. Adenosine 5′-triphosphate    (ATP) supplements are not orally bioavailable: a randomized,    placebocontrolled cross-over trial in healthy humans. J. Int. Soc.    Sports Nutr. 9: 16, 2012.-   34. Coolen, E. J., Arts, I. C., Bekers, O., Vervaet, C., Bast, A. &    Dagnelie, P. C. Oral bioavailability of ATP after prolonged    administration. Br. J. Nutr. 105: 357-366, 2011.-   35. Synnestvedt, K., Furuta, G. T., Comerford, K. M., Louis, N.,    Karhausen, J., Eltzschig, H. K., Hansen, K. R., Thompson, L. F. &    Colgan, S. P. Ecto-5′-nucleotidase (CD73) regulation by    hypoxia-inducible factor-1 mediates permeability changes in    intestinal epithelia. J Clin. Invest 110: 993-1002, 2002.-   36. Kraemer, W. J., Hatfield, D. L., Volek, J. S., Fragala, M. S.,    Vingren, J. L., Anderson, J. M., Spiering, B. A., Thomas, G. A.,    Ho, J. Y. et al. Effects of Amino Acids Supplement on Physiological    Adaptations to Resistance Training. Med. Sci. Sports Exerc. 41:    1111-1121, 2009.-   37. Monteiro, A. G., Aoki, M. S., Evangelista, A. L., Alveno, D. A.,    Monteiro, G. A., Picarro, I. C. & Ugrinowitsch, C. Nonlinear    periodization maximizes strength gains in split resistance training    routines. J Strength. Cond. Res. 23: 1321-1326, 2009.-   38. Laurent, C. M., Green, J. M., Bishop, P. A., Sjokvist, J.,    Schumacker, R. E., Richardson, M. T. & Curtner-Smith, M. A practical    approach to monitoring recovery: development of a perceived recovery    status scale. J Strength. Cond. Res. 25: 620-628, 2011.-   39. Rathmacher, J. A., Link, G. A., Flakoll, P. J. & Nissen, S. L.    Gas chromatographic-mass spectrometric analysis of stable isotopes    of 3-methylhistidine in biological fluids: application to plasma    kinetics in vivo. Biol. Mass Spectrom. 21: 560-566, 1992.-   40. Barnes J N, Trombold J R, Dhindsa M, Lin H F, and Tanaka H.    Arterial stiffening following eccentric exercise-induced muscle    damage. Journal of applied physiology 109: 1102-1108, 2010.-   41. Cormie P, McGuigan M R, and Newton R U. Developing maximal    neuromuscular power: Part 1—biological basis of maximal power    production. Sports medicine 41: 17-38, 2011.-   42. Cormie P, McGuigan M R, and Newton R U. Developing maximal    neuromuscular power: part 2—training considerations for improving    maximal power production. Sports medicine 41: 125-146, 2011.-   43. Dufour S P, Patel R P, Brandon A, Teng X, Pearson J, Barker H,    Ali L, Yuen A H, Smolenski R T, and Gonzalez-Alonso J.    Erythrocyte-dependent regulation of human skeletal muscle blood    flow: role of varied oxyhemoglobin and exercise on nitrite,    S-nitrosohemoglobin, and ATP. American journal of physiology Heart    and circulatory physiology 299: H1936-1946, 2010.-   44. Gilbert G and Lees A. Changes in the force development    characteristics of muscle following repeated maximum force and power    exercise. Ergonomics 48: 1576-1584, 2005.-   45. Gonzalez-Alonso J. ATP as a mediator of erythrocyte-dependent    regulation of skeletal muscle blood flow and oxygen delivery in    humans. The Journal of physiology 590: 5001-5013, 2012.-   46. Gonzalez-Alonso J, Mortensen S P, Dawson E A, Secher N H, and    Damsgaard R. Erythrocytes and the regulation of human skeletal    muscle blood flow and oxygen delivery: role of erythrocyte count and    oxygenation state of haemoglobin. The Journal of physiology 572:    295-305, 2006.-   47. Gonzalez-Alonso J, Mortensen S P, Jeppesen T D, Ali L, Barker H,    Damsgaard R, Secher N H, Dawson E A, and Dufour S P. Haemodynamic    responses to exercise, ATP infusion and thigh compression in humans:    insight into the role of muscle mechanisms on cardiovascular    function. The Journal of physiology 586: 2405-2417, 2008.-   48. Halson S L and Jeukendrup A E. Does overtraining exist? An    analysis of overreaching and overtraining research. Sports medicine    34: 967-981, 2004.-   49. Hunga W, Liub T-H, Chenc C-Y, and Chang C-K. Effect of    [beta]-hydroxy-[beta]-methylbutyrate Supplementation During Energy    Restriction in Female Judo Athletes. Journal of Exercise Science and    Fitness 8: 50-53, 2010.-   50. Jowko E, Ostaszewski P, Jank M, Sacharuk J, Zieniewicz A,    Wilczak J, and Nissen S. Creatine and    beta-hydroxy-beta-methylbutyrate (HMB) additively increase lean body    mass and muscle strength during a weight-training program. Nutrition    17: 558-566, 2001.-   51. Kraemer W J and Ratamess N A. Fundamentals of resistance    training: progression and exercise prescription. Med Sci Sports    Exerc 36: 674-688, 2004.-   52. Rathmacher J A, Fuller J C, Jr., Baier S M, Abumrad N N, Angus H    F, and Sharp R L. Adenosine-5′-triphosphate (ATP) supplementation    improves low peak muscle torque and torque fatigue during repeated    high intensity exercise sets. Journal of the International Society    of Sports Nutrition 9: 48, 2012.-   53. Robbins D W and Docherty D. Effect of loading on enhancement of    power performance over three consecutive trials. Journal of strength    and conditioning research/National Strength & Conditioning    Association 19: 898-902, 2005.-   54. Sprague R S, Bowles E A, Achilleus D, Stephenson A H, Ellis C G,    and Ellsworth M L. A selective phosphodiesterase 3 inhibitor rescues    low PO2-induced ATP release from erythrocytes of humans with type 2    diabetes: implication for vascular control. American journal of    physiology Heart and circulatory physiology 301: H2466-2472, 2011.-   55. Thomson J S, Watson P E, and Rowlands D S. Effects of nine weeks    of beta-hydroxy-beta-methylbutyrate supplementation on strength and    body composition in resistance trained men. Journal of strength and    conditioning research/National Strength & Conditioning Association    23: 827-835, 2009.-   56. Trautmann A. Extracellular ATP in the immune system: more than    just a “danger signal”. Science signaling 2: pe6, 2009.-   57. van Someren K A, Edwards A J, and Howatson G. Supplementation    with beta-hydroxy-beta-methylbutyrate (HMB) and alpha-ketoisocaproic    acid (KIC) reduces signs and symptoms of exercise-induced muscle    damage in man. International journal of sport nutrition and exercise    metabolism 15: 413-424, 2005.-   58. Wilkinson D J, Hossain T, Hill D S, Phillips B E, Crossland H,    Williams J, Loughna P, Churchward-Venne T A, Breen L, Phillips S M,    Etheridge T, Rathmacher J A, Smith K, Szewczyk N J, and Atherton    P J. Effects of Leucine and its metabolite,    beta-hydroxy-beta-methylbutyrate (HMB) on human skeletal muscle    protein metabolism. The Journal of physiology, 2013.-   59. Wilson G J, Wilson J M, and Manninen A H. Effects of    beta-hydroxy-beta-methylbutyrate (HMB) on exercise performance and    body composition across varying levels of age, sex, and training    experience: A review. Nutr Metab (Lond) 5: 1, 2008.-   60. Wilson J M, Duncan N M, Marin P J, Brown L E, Loenneke J P,    Wilson S M, Jo E, Lowery R P, and Ugrinowitsch C. Meta-Analysis of    Post Activation Potentiation and Power: Effects of Conditioning    Activity, Volume, Gender, Rest Periods, and Training Status. Journal    of strength and conditioning research/National Strength &    Conditioning Association, 2012.-   61. Wilson J M, Lowery R P, Joy J M, Walters J, Baier S, Fuller J C,    Jr., Stout J, Norton L, Sikorski E M, Wilson S M-C, Duncan N, Zanchi    N, and Rathmacher J. β-Hydroxy-β-Methylbutyrate Free Acid Reduces    Markers of Exercise Induced Muscle Damage and Improves Recovery in    Resistance Trained Men. British Journal of Nutrition In Press.

The invention claimed is:
 1. A composition comprising a synergisticcombination of from about 0.5 g to about 30 g ofβ-hydroxy-β-methylbutyric acid (HMB) and from about 10 mg to about 80 gof adenosine triphosphate (ATP).
 2. A method for providing a benefit toan animal in need thereof selected from the list consisting ofincreasing strength, increasing power, improving muscle mass, andlessening declines in performance characteristic of overreaching byadministering to the animal a composition comprising a synergisticcombination of an effective amount of HMB and ATP, wherein the amount ofHMB administered is from about 0.5 to about 30 g and the amount of ATPadministered is from about 10 mg to about 80 g of ATP.
 3. The method ofclaim 2, wherein the HMB administered is HMB-acid.
 4. The method ofclaim 2, wherein the HMB administered is a salt.
 5. The method of claim2, wherein the step of administering is selected from the groupconsisting of oral, parenteral, sublingual, topical, transdermal,intramuscular, and inhalation.
 6. The method of claim 5, wherein theoral administration comprises a delivery form selected from the groupconsisting of tablet, capsule, powder, granule, microgranule, pellet,soft-gel, controlled-release form, liquid, solution, elixir, syrup,suspension, emulsion and magma.
 7. A method for increasing strength ofan animal in need thereof comprising the steps of administering to saidanimal a synergistic combination of from about 0.5 to about 30 g HMB andfrom about 10 mg to about 80 g ATP i, wherein upon said administrationof HMB and ATP to the animal, said strength is increased.
 8. A methodfor increasing power of an animal in need thereof comprising the stepsof administering to said animal a synergistic combination of from about0.5 to about 30 g HMB and from about 10 mg to about 80 g ATP , whereinupon said administration of HMB and ATP to the animal, said power isincreased.
 9. A method for improving the muscle mass of an animal inneed thereof comprising the steps of administering to said animal asynergistic combination of from about 0.5 to about 30 g HMB and fromabout 10 mg to about 80 g ATP, wherein upon said administration of HMBand ATP to the animal, said muscle mass is improved.
 10. A method forlessening declines in performance characteristic of overreaching for ananimal in need thereof comprising the steps of administering to saidanimal a synergistic combination of from about 0.5 to about 30 g HMB andfrom about 10 mg to about 80 g ATP, wherein upon said administration ofHMB and ATP to the animal, said declines in performance are lessened.11. A composition comprising a combination of from about 0.5 g to about30 g of β-hydroxy-β-methylbutyric acid (HMB) and from about 10 mg toabout 80 g of adenosine triphosphate (ATP) for use in providing abenefit to an animal in need thereof selected from the list consistingof increasing strength, increasing power, improving muscle mass, andlessening declines in performance characteristic of overreaching. 12.The composition of claim 11, wherein the HMB is a salt, HMB-acid, alactone or an ester.
 13. A method for providing a benefit to an animalin need thereof selected from the list consisting of increasingstrength, increasing power, improving muscle mass, and lesseningdeclines in performance characteristic of overreaching by administeringto the animal a composition comprising a combination from about 0.5grams to about 30 grams of HMB and from about 10 mg to about 80 mg ofATP.
 14. The method of claim 13, wherein the HMB administered is a salt,HMB-acid, a lactone or an ester.
 15. The method of claim 13, wherein thestep of administering is selected from the group consisting of oral,parenteral, sublingual, topical, transdermal, intramuscular, andinhalation.
 16. The method of claim 15, wherein the oral administrationcomprises a delivery form selected from the group consisting of tablet,capsule, powder, granule, microgranule, pellet, soft-gel,controlled-release form, liquid, solution, elixir, syrup, suspension,emulsion and magma.
 17. The composition of claim 1, wherein the HMB is asalt, HMB-acid, a lactone or an ester.
 18. The method of claim 2,wherein the HMB is a lactone or an ester.